Translation of mRNAs normally terminates at the first in-frame stop codon encountered by the ribosome; however, in some circumstances, translation can continue beyond the stop codon to a downstream stop codon, generating a novel protein with a C-terminal polypeptide extension. Such translational readthrough (TR) events are best understood in viruses, and evidence for TR of vertebrate mRNAs has been accumulating in the last two years. However, essentially nothing is known about the molecular mechanisms that regulate TR in vertebrate mRNAs. We have recently reported that protein-driven, programmed TR of human VEGFA (vascular endothelial growth factor-A) mRNA in endothelial cells (EC) generates a novel isoform of VEGF-A, a secreted angiogenic mitogen that regulates blood vessel formation. Readthrough generates a protein termed VEGF-Ax (x for extended) containing a 22-amino acid C-terminus extension. Remarkably, the RNA sequence that encodes the 22-amino acid extension also functions as a recoding cis-acting element (Ax element) essential for efficient TR. Readthrough is stimulated by interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1 with the Ax element. Recent preliminary studies show an ensemble of proteins, including hnRNP L, DDX5, and HuR, also bind the Ax element. VEGF-Ax expression is down-regulated during C2C12 myoblast differentiation, but is remarkably efficient in human platelets. Based on these initial studies, we propose the following specific hypothesis: Assembly of a multiprotein complex on the structured VEGFA mRNA Ax element interacts with the ribosome or release factors to recode the canonical stop codon, regulating cell- and context-dependent TR and VEGF-Ax generation. We will test this hypothesis by pursuing three Specific Aims: Aim 1. Identify Ax element-binding trans-acting factors (AxE-TAF) and their role in TR of VEGFA mRNA in EC. We will identify and validate AxE-TAFs, and determine their interactions with each other, their affinities for the Ax element, and their influence on TR efficiency. We will also investigate the role of post-translational modifications (PTM) of these factors on their interactions and cell localization. Aim 2. Investigate determinants of cell- and context-dependent TR efficiency of VEGFA mRNA. We will investigate the mechanisms underlying suppressed VEGF-Ax production during C2C12 myoblast differentiation, and extraordinarily efficient VEGF-Ax expression in human platelets. We will determine the influence of localization, expression, and PTM of regulatory trans-acting factors on assembly of the readthrough complex, binding to the Ax element, and readthrough efficiency. Aim 3. Determine molecular mechanisms regulating translational readthrough of VEGFA mRNA. We will determine the folding structure of the Ax RNA element and its functional regions and residues. We will test the hypothesis that readthrough is promoted by the interaction of AxE-TAFs with ribosomes or ribosomal release factors, and identify the tRNA responsible for Ser insertion. TR has recently emerged as a mechanism of expansion of the human proteome. Successful completion of the proposed studies will not only elucidate mechanisms of readthrough of VEGFA mRNA, but also will provide a foundation for investigation of mechanisms regulating TR of other mRNAs and systems.